Evaluation of growth, antioxidant status, hepatic enzymes and immunity of Nanoselenium-Fed Cirrhinus mrigala

This study was conducted to investigate the effects of selenium nanoparticle (Se-NP) supplementation on the growth performance, carcass composition, antioxidant status, hepatic enzyme activities, and immunity of Cirrhinus mrigala. For this purpose, fish with an average initial weight of 7.44 ± 0.04 g were fed five experimental diets containing 0 (control), 0.25, 0.5, 1, and 2 mg kg-1 Se-NPs diets for 90 days. The analysed selenium (Se) contents of the diets were 0.35, 0.64, 0.92, 1.43, and 2.39 mg kg-1. Twenty five fish were randomly distributed in each of 5 aquarium (36 × 23.7 × 24.3 inches) in triplicate. The results showed that supplementation with Se up to 0.92 mg/kg significantly increased (p<0.05) weight gain, weight gain% (WG%), and specific growth rate (SGR) by 34%, 33%, and 16%, respectively, compared to the control diet. Dietary Se concentrations up to 0.92 mg/kg significantly increased the crude protein and crude fat and reduced (p<0.05) the moisture content as compared to the control group. Fish fed 0.92 mg kg-1 Se had significantly lower malondialdehyde (MDA) contents and higher activities of catalase, superoxide dismutase, and glutathione peroxidase in liver and serum as compared to other experimental diets. Moreover, a significant increase (p<0.05) in the level of serum immunoglobulin and lysozyme (LYZ) activity was recorded in fish fed 0.92 mg/kg Se diet. Moreover, the highest (p<0.05) values of aspartate transaminase (AST) and alanine transaminase (ALT) were recorded in fish fed 2.39 mg/kg Se level. However, serum alkaline phosphatase (ALP) activity remained unaffected by dietary treatment. Broken-line regression analysis indicated that 0.83 mg/kg Se is required for the optimum growth performance of C. mrigala.


Introduction
Aquaculture has immense importance in providing the global population with high-quality proteins and future food security.The growing population has exerted great pressure on aquaculture and aquaculture practices have moved towards intensification to meet the demand [1].However, culturing fish at a high stocking density results in frequent collisions, competition for food and space, and deterioration of water quality.These factors contribute to stress conditions that negatively correlate with the growth and health status of fish [2].Oxidative stress increases the generation of free radicals, which damages the membranes of cells and leads to depressed growth, immunity, and ultimately mortality.The sustainability of aquaculture has been linked with strategies to reduce stress conditions to boost production and profitability [3].Several strategies, such as antibiotics, vaccines, and other synthetic chemicals, are being employed, which poses serious health concerns [4].Dietary manipulation with micronutrients has been suggested as the most effective and safest method to enhance the health status and growth of fish [5].
Selenium is an indispensable trace element required in small quantity that acts as a great immune stimulator and antioxidant [6].In addition to nutritional requirement, Se is also required for the synthesis of selenoproteins such as glutathione peroxidase (GPx), which is an important antioxidant enzyme that acts as the first line of defense against free radicals [7].However, selenium acts as both an immune stimulator and toxic with a minute difference in dose.According to a report, 0.2-12 mg kg -1 Se is required to supplement the diet of different fish species [8].Deficiency of Se caused muscular dystrophy, swollen endoplasmic reticulum, and myocardial degeneration in fish [9].However, high Se levels induced toxic effects on fish health and resulted in teratogenic deformities of fish organs, tissue destruction, nephrocalcinosis, and stunted growth [10].Moreover, the chemical form of dietary-supplemented Se in feed formulations is an important consideration [10].Previous studies have shown that organic forms of Se have a better ability to accumulate and be digested in fish bodies than inactive forms [11].However, in some cases, an organic form of Se has less functionality due to its high pH, which in turn reduces its solubility in water [12].
Nanotechnology is emerging as a novel and safe approach to enhance nutrient absorption and utilization due to the very small size of particles.It has been confirmed that dietary Se nanoparticle (Se-NP) supplementation is more efficient in improving the immune response of fish than organic or inorganic forms [13].These Se-NPs have gained attention in aquafeeds due to their low toxicity, higher bioactivity, biocompatibility, strong adsorption properties, and high chemical consistency [14].In crucian carp, supplementation with selenium nanoparticles (Se-NPs) had more positive impacts on the muscle Se content than the organic form [15]. Previous studies have also shown that supplementation with Se-NPs improved the growth performance of different fish species [7,16,17].
Cirrhinus mrigala (mori) is an important fish of the Indian subcontinent.It contributes 67% of total freshwater aquaculture production with Catla catla and Labeo rohita [18].This species is considered a potential candidate for the polyculture system in the Indo-Pak region.Hence, the formation of nutritionally balanced feed is an absolute need of the moment for intensive farming practices of this species.Therefore, this study was undertaken to determine the optimum level of Se for Cirrhinus mrigala juveniles.

Ethical approval
This study was conducted after approval (No.DR/237) from the Ethical Review Committee of the University of Veterinary and Animal Sciences, Lahore, according to the standard guidelines of the institute.

Diet formulation
Se-NPs (average particle size: <80 nm; purity>99%; Nanochemazone, Canada) were used as a source of Se.Five experimental diets were formulated containing 0, 0.25, 0.5, 1, and 2 mg/kg Se-NPs (Table 1).Feed ingredients were air-dried, crushed (KENWOOD, AT284), screened (0.05 mm), and thoroughly mixed with Se-NPs and fish oil.Distilled water was used for the formation of stiff dough, which was pelleted through meat mincer (ANEX, AG3060).Pellets (1 mm) were shade-dried and stored in airtight labelled containers for the feeding trial.

Experimental fish and feeding trial
A total of 375 C. mrigala juveniles were procured from Arain Fish Farm (local farm located 10 km from laboratory) in Pattoki, Pakistan.Fish were transported in polyethlene bags following the standard protocol described by Berka [19].Upon arrival in the laboratory, fish were bathed in anesthetic solution (5 g/L KMnO 4 ) to remove any parasites.Then, fish were acclimatized in a circular tank (2000 L capacity) with a 0.3 L/min water exchange rate and continuous aeration for 15 day [20].Fish were fed a control diet twice daily @3% body weight ratio during acclimatization.At the beginning of the experiment, the initial body weight (7.44 ± 0.04 g) of the fish was recorded, and the fish were randomly distributed into 15 aquaria (36 × 23.7 × 24.3 inches) at a density of 25 fish per aquarium.The experimental diets were given to fish @3% body weight ratio twice daily for 90 days.After 3 hours of feeding, uneaten feed was collected from each tank to estimate feed intake (FI).Fish were batch weighed every fortnight, and feeding amount was adjusted accordingly.The water quality was maintained by replacing 50% of the tank water with fresh water daily [21].
The water quality parameters, such as dissolved oxygen, pH, and temperature, were monitored regularly throughout the feeding trial, and their mean values were 7.6 ± 0.3 mg per liter, 7.2 ± 0.2 and 28.5 ± 0.7˚C, respectively.

Sample collection
At the end of 90 days of feeding trial, fish were deprived of feed for 24 hours and then fish were anaesthetized with 25 mg/L tricaine methanesulfonate [22].For proximate composition analysis, five fish were taken at random from each replicate.Another 5 fish were taken from each replicate and were dissected to collect liver samples for the analysis of antioxidant enzymes activitiesand lipid peroxidation assays.Blood samples were collected from 5 fish of each replicates by puncture the caudal vein using 3 mL sterile syringe.Blood samples were immediately transported in laboratory for serum separation.

Growth performance and feed utilization
At experiment termination, fish were individually weighed to determine the weight gain (WG), specific growth rate (SGR), feed intake (FI), feed conversion ratio (FCR), and survival rate (SR) using the following standard equations.

Proximate analysis
The AOAC [23] procedure was used for the estimation of proximate composition.Briefly, moisture was determined through the oven-drying method, and crude protein (N×6.25) was estimated by Kjeldahl's method.The Soxhlet method was used for the determination of crude fat.Crude ash was estimated by placing the samples at 660˚C in a muffle furnace.Atomic absorption spectroscopy (Hitachi ZA3000, Chiyoda, Tokyo, Japan) was used for the estimation of Se in diets [24].

Antioxidant enzyme activities and lipid peroxidation
Hepatic samples from each replicate were pooled and homogenized with phosphate buffer to obtain the extract.After centrifugation of the homogenized samples, the supernatant layer was separated and stored for antioxidant enzyme assays.Glutathione peroxidase (GPx) activity was determined at A 470 nm by measuring its ability to decrease H 2 O 2 concentration [25].Catalase (CAT) activity was estimated by its potential to reduce H 2 O 2 concentration at 240 nm [26].
The activity of superoxide dismutase (SOD) was estimated by its capacity to prevent photoreduction of nitroblue tetrazole [27].Malondialdehyde (MDA) content was measured colorimetrically by following Gatta, Pirini [28].Serum antioxidant enzyme activities and MDA contents were also determined using same methods.

Immune parameters
Lysozyme (LYZ) activity was estimated by the lysis of Staphylococcus aureus by following the standard protocol of Ellis [29].Total immunoglobulin was determined by following the procedure of [30].
Total Immunoglobulin ¼ total ptotein from sample À protein content in supernatant

Serum hepatic enzymes
The commercial kit method (CHEMELEX, S. A, Pol.Ind. Can Castells, Spain) was used to determine the activities of hepatic enzymes, including alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST), in serum.

Statistical analysis
The obtained data were subjected to one-way analysis of variance, and Duncan's multiple range test was applied to compare the means in SPSS (version 23).To determine the optimum dietary requirement of Se, broken-line regression analysis was used.

Growth performance and feed utilization
The supplementation of Se up to 0.92 mg/kg significantly increased (p<0.05)FBW, WG, WG %, and SGR by 25%, 34%, 33%, and 16%, respectively, compared with the control diet.However, a slight decline (p<0.05) in these parameters was noted with a further increase in Se concentration (Table 2).The best values (p<0.05) of FI and FCR were recorded at the 0.92 mg/kg Se level.No mortality was recorded throughout the trial.Based on broken-line regression analysis, supplementation with 0.83 mg/kg Se was recommended for the maximum WG% of C. mrigala (Fig 1).

Carcass composition
Supplementation with Se significantly improved (p<0.05) the whole-body crude protein and crude fat while reducing (p<0.05) the moisture content of C. mrigala.However, crude ash remained unaffected by Se supplementation (Table 3).Fish fed the diets with 0.92 mg/kg and 1.43 mg/kg Se had significantly higher (p<0.05)whole-body crude protein, followed by the fish fed 2.39 mg/kg Se, then 0.64 mg/kg Se and finally the control diet.The crude fat content increased (p<0.05) from 4.30% to 5.33% with increasing Se supplementation levels up to 0.92 mg/kg and decreased (p<0.05)thereafter.

Liver and serum antioxidant status and lipid peroxidation
The antioxidant response of C. mrigala in response to Se supplementation is summarized in Table 4. SOD activities responded positively (p<0.05) with higher Se concentrations up to 0.92 mg/kg diet.However, a slight decline (p<0.05) in its values was observed thereafter.CAT activities tended to increase (p<0.05) from 70.52 to 75.45 U/g prot up to the 0.92 mg/kg Se level and decreased significantly (p<0.05)beyond this level.Similarly, GPx activities improved (p<0.05) with Se levels, peaking at 0.92 mg/kg and declining (p<0.05)thereafter.MDA  contents were reduced (p<0.05)from 3.67 to 3.11 mg/g prot with higher Se up to 0.92 mg/kg diet.However, a gradual increase in its content was recorded at higher Se levels.

Nonspecific immune response
Fish fed the Se-containing diets showed significantly elevated (p<0.05)lysozyme activity compared with those fed the control diet (Table 5).Furthermore, immunoglobulin increased (p<0.05) from 0.74 to 1.02 g/g pro as Se increased from 0.35 to 0.92 mg/kg diet and then declined significantly at the 1.43 and 2.39 mg/kg levels.

Serum hepatic enzymes
Fish fed the diets containing � 1.43 Se exhibited a nonsignificant difference in ALT values compared to the fish given the control diet.However, the highest values (p<0.05) of ALT were observed at the 2.39 mg/kg level.Similarly, a subsequent increase (p<0.05) in AST values was recorded as Se increased from 0.92 to 2.39 mg/kg diet.However, ALP remained unaffected by dietary treatment (Table 6).

Discussion
Feed is the most important component of aquaculture which consists of macro and micronutrients that are essential for the optimum growth performance and health status of fish [31].Selenium is an essential mineral which plays various physiological functions in fish [32].Furthermore, optimum level of Se is also important as under or over supplementation results in depressed growth performance or various physiological dysfunctions [32].Fish require about  [10,16,[33][34][35][36][37][38][39].In this study, 0.83 mg/kg Se was calculated as optimum requirement of C. mrigala based on WG% (Fig 1).In this study, dietary supplementation of Se-NPs enhanced the growth performance and feed utilization in C. mrigala.Currenlty, no study has reported the optimum dietary Se-NPs for C. mrigala.Se has been shown to enhance growth hormone (thyroid) production in fish, contributing to improved growth performance [40].Se interacts with the deiodinase enzyme, a key component in the regulation of thyroid hormones [41].In fish, as in other vertebrates, the pituitary gland plays a crucial role in secreting thyroid hormones, which subsequently stimulate the production of growth hormones [42].Moreover, Se enhances intracellular protein content in the epithelial cells of the intestine, which in turn improves the feed utilization and growth of fish [43].Furthermore, the functional role of Se as a coenzyme in the synthesis of digestive enzymes improves the digestibility and absorption of nutrients in the intestine, resulting in better growth performance of fish [44].
The proximate composition of fish, encompassing parameters such as moisture, protein, lipid, and ash content, is of paramount importance in multiple contexts.It serves as a critical metric for assessing the nutritional value of fish [45].In the current study, supplementation of Se-NPs significantly enhanced crude protein and crude lipid content in whole body of C. mrigala which coincides with previous studies [17,35,39,46].This increase in protein and lipids in whole body can be explained as Se enhances the secretion of digestive enzymes which boost the digestion of proteins and lipids [44].Furthermore, improvement in intestine health ultimate results in better absorption of nutrients which are stored in body [43].Furthermore, Dietary Se participates in building selenoproteins that enhance proteins' synthesis in the GIT and enhance protein accumulation in the tissues.
Aquaculture, especially intensive culture system, is prone to various stressors which lead oxidative stress which ultimately reduce growth performance and negatively impact the health status of fish [5].During stress condition, free radicals are produced and fish have a natural antioxidant mechanism which neutralizes these free radicals [47].However, when the production of free radicals exceeds the neutralizing capacity of antioxidant system, then these free radicals attack the lipids in cell membranes and produce malondialdehyde [43].The main components of antioxidant defence system are catalase, superoxide dismutase and glutathione peroxidase which scavange free radicals.In current study, dietary supplementation of Se-NPs enhanced the antioxidant potential in C. mrigala which coincides with the previous studies [16,33,[35][36][37]48].This can be explained as Se has a key role in synthesis of glutathione peroxidase enzyme.This enzyme with the help of CAT and SOD neutralizes the free radicals ultimately protecting fish from deleterious effects of free radicals.Furthermore, Se-NPs have also been reported to enhance the gene expression of GPx [42].
Immunoglobulins are vital components of the adaptive immune system in fish, serving key roles in pathogen defense and immune regulation [49].They are primarily involved in the recognition and neutralization of specific antigens, thereby preventing infections and contributing to the organism's overall immune response [49,50].Upon antigen binding, immunoglobulins can activate complement proteins and phagocytic cells, which work to eliminate pathogens from the host [50].It was observed that the serum immunoglobulin of C. mrigala increased significantly with Se-NP supplementation which is in line with previous studies [35,40].This might be due to stimulation of GPx in those cells, which in turn activates and protects B lymphocytes, resulting in a better immune response [51].
Lysozymes complement the function of immunoglobulins in fish immune defense.These enzymes are part of the innate immune system and are known for their ability to break down bacterial cell walls by hydrolyzing the peptidoglycan layer [52].This bacteriolytic activity is crucial for protecting fish against bacterial infections, particularly in environments where they are exposed to a wide variety of microbial threats.Lysozymes are found in various tissues and body fluids of fish, including mucus, blood, and internal organs, providing a broad-spectrum antimicrobial barrier [53].Supplementation with Se had a significant positive impact on lysozyme activities which is in accordance to previous studies [22,36,37,40].This enhancement in lysozyme activity might be due to well-known immunomodulatory role of Se in the immune system via contributing to the proliferation, differentiation, and regulation of the immune cells (e.g., lymphocytes and neutrophils) [54].
ALP, ALT, and AST are important enzymes involved in metabolism.A significant increase in ALT and AST levels was recorded at the 2.39 mg/kg level in this study, indicating that an overdose of Se may cause toxic effects on the liver health of fish.It was reported that Se mainly accumulates in the liver of fish, and a high concentration of Se may lead to the degeneration of liver tissues [55].Similarly, Ashouri, Keyvanshokooh [16] noted the highest levels of serum ALT and AST at the 2 mg/kg Se-NP level in common carp.In contrast, Yu, Zhang [56] reported a decreasing trend in AST and ALT values with increasing levels of Se-NPs in Ctenopharyngodon idella, whereas no significant effects of Se-NPs on AST and ALT levels were observed in Dicentrarchus labrax [22].The improvements in hepatic enzymes activities with Se supplementation can be associated with the enhancement of antioxidant status, immune response and overall health of fish as described previously.

Conclusion
The findings of the study showed that supplementation with up to 0.92 mg/kg Se in the form of NPs significantly improved the growth performance, feed utilization, whole-body composition, antioxidant capacity, and immunity of C. mrigala.Based on broken-line regression analysis, 0.83 mg/kg Se is recommended for the optimum performance of C. mrigala.To date, very few studies have investigated the effect of Se-NPs on carp.Therefore, more concerning efforts should be devoted to this regard.

Table 2 . Growth performance and feed utilization of Cirrhinus mrigala given experimental diets.
Data are shown as the mean ± SD of triplicate tanks.Means having dissimilar letters within a row indicate significant difference (p < 0.05).https://doi.org/10.1371/journal.pone.0308761.t002

Table 5 . Nonspecific immune parameters of Cirrhinus mrigala given experimental diets.
.2-12 mg/kg Se in diet depending on various factors such as specie, size, feed formulation and culture conditions Data are shown as the mean ± SD of triplicate tanks.Means with dissimilar letters indicate significant differences (p < 0.05).https://doi.org/10.1371/journal.pone.0308761.t0050